Fig 2: The TERT protein levels in the ovarian follicles from primary to antral stages, obtained from adult (6-week-old) and aged (52-week-old) groups. In this analysis, we used primary (n = 60 and 46), secondary (n = 77 and 58), preantral (n = 59 and 64), and antral (n = 56 and 38) follicles from 14 adult and 9 aged mice, respectively. (A) Western blotting (WB) results showing bands corresponding to the TERT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins in the isolated follicles. GAPDH (in the molecular weight of 35 kDa) was used as an internal loading control. All the follicles either from adult or aged groups possessed the TERT protein (in the molecular weight of 127 kDa) at different levels. The bands produced via WB were analyzed using the ImageJ software program version 2.0 (bundled with 64-bit Java 1.8.0_172; NIH, Bethesda, Maryland, USA, URL link: https://imagej.nih.gov/ij/download), and the results were presented as a column chart. PF primary follicles, SF secondary follicles, PreF preantral follicles, AF antral follicles. (B) The relative TERT protein levels in the follicles from the adult group. The highest and lowest TERT protein levels were observed in the primary (P < 0.01) and secondary (P < 0.01) follicles, respectively. It should be noted that there were statistical significances among all follicle types (##P < 0.01). (C) The relative TERT protein levels in the follicles from the aged group. It progressively decreased from primary to antral follicles except for the secondary follicles, where a marked reduction was observed (P < 0.01). It is worth noting that there were statistical significances among all follicle types (##P < 0.01). (D) The comparison of relative TERT protein levels in the follicles between adult and aged groups. In the aged group, we detected lower TERT protein levels in the primary, preantral, and antral follicles (P < 0.01), but higher levels in the secondary follicles (P < 0.01) when compared to those of their adult counterparts. There were statistical significances among all follicle types (**P < 0.01). Here, the statistical significances in B and C were evaluated using one-way ANOVA test and the Student–Newman–Keuls post hoc test (denoted by ##); the student’s t-test (denoted by **) was used in D. The data obtained are presented as means ± standard deviations (SDs; arising from two technical repeats).

Fig 3: The Tert, Terc, Trf1, Trf2, and Pot1a gene expressions were evaluated in the ovarian follicles from primary to antral stages, obtained from adult (6-week-old) and aged (52-week-old) groups. Expression levels of these genes were normalized against those of ß-Actin. It is important to note that expression level of each gene in the primary follicles of the adult group was set to 1 in all analyses. In this analysis, we used primary (n = 32 and 25), secondary (n = 45 and 50), preantral (n = 35 and 36), and antral (n = 31 and 24) follicles from 14 adult and 9 aged mice, respectively. (A) The relative Tert mRNA levels in the follicles. Although the primary and antral follicles of the aged group displayed lower Tert mRNA levels compared to their adult counterparts (P < 0.001), the secondary follicles of the aged group exhibited a higher profile than those from adult group (P < 0.001). ###P < 0.001 for the primary follicles versus the secondary and preantral follicles, and the antral follicles versus the preantral follicles in the adult group. ###P < 0.001 for the secondary follicles versus the remaining follicles in the aged group, as well as for the antral follicles versus the primary and secondary follicles. ***P < 0.001 for the follicles between adult and aged groups. (B) The relative Terc RNA levels in the follicles. Only the secondary follicles from the aged group exhibited a higher Terc RNA level (P < 0.001); however, other follicle types displayed lower profiles compared to their adult counterparts (P < 0.001). ###P < 0.001 for the secondary follicles versus the remaining follicles in the adult group. ###P < 0.001 for the secondary follicles versus the remaining follicles, as well as for the preantral follicles versus the secondary follicles in the aged group. ***P < 0.001 for the follicles between adult and aged groups. (C) The relative Trf1 mRNA levels in the follicles. The secondary follicles from the aged group had significantly higher Trf1 mRNA levels (P < 0.001), whereas the preantral and antral follicles exhibited lower levels, compared to their respective adult counterparts (P < 0.001). ###P < 0.001 for the preantral follicles versus the primary and secondary follicles, ##P < 0.01 for the antral follicles versus the remaining follicles in the adult group. In the aged group, ###P < 0.001 for the secondary and antral follicles versus the primary follicles, and for the preantral follicles versus the remaining follicles. ***P < 0.001 for the follicles between adult and aged groups. (D) The relative Trf2 mRNA levels in the follicles. The secondary follicles from the aged group displayed higher Trf2 mRNA level compared to those of the adult group (P < 0.001); this occurrence was reversed in the antral follicles (P < 0.001). ###P < 0.001 for the antral follicles versus the remaining ones, and for the preantral follicles versus the primary and secondary follicles in the adult group. In the aged group, ###P < 0.001 for the secondary follicles versus the remaining ones, and for the preantral follicles versus the primary follicles. ***P < 0.001 for the follicles between adult and aged groups. (E) The relative Pot1a mRNA levels in the follicles. The aged group exhibited higher Pot1a mRNA levels in the primary and secondary follicles, but lower level in the antral follicles, compared to their adult counterparts (P < 0.001). ###P < 0.001 for the antral follicles versus the remaining ones, and for the preantral follicles versus the primary and secondary follicles in the adult group. In the aged group, ###P < 0.001 for the antral follicles versus the remaining ones, and ##P < 0.01 for the preantral follicles versus the primary and secondary follicles. ***P < 0.001 for the follicles between adult and aged groups. Here, the data obtained from the follicles in each group and between the groups were analyzed using one-way ANOVA and the Holm-Sidak post hoc test (denoted by ## or ###), and using the student’s t-test (denoted by ***), respectively. We presented the values as means ± standard deviations (SDs; arising from three technical repeats).

Fig 4: TERT protein level in (A) the cerebellum; and (B) the cortex. *P<0.05. TERT, telomerase reverse transcriptase.

Fig 5: Further characterization of the FH+/– cells. (A) Microscopic images of WT and FH+/– cells at the 61st passage (200×). (B) Representative ultrastructure images of WT and FH+/– cells at the 55th passage evaluated by electron microscopy (a,d:2000×; b,e:4000×; c.f:8000×), where a larger cell body with plenty of rough endoplasmic reticulum, mitochondria and ribosomes is shown in the FH+/– cells, but the WT fibroblasts show fewer mitochondria and ribosomes and deformed and less condensed rough endoplasmic reticulum. (C) Representative immunofluorescence images of the expression of p53, p21 and p16 (200×). Western blot for p53, p21, p16 and TERT in WT and FH+/– cells and the corresponding histograms are shown in (D) and (E), respectively. (F) SA-ß-Gal detection . (G) FH+/– single cell cloning images captured with the Incucyte Zoom System for cells at the 84th passage. (H) The karyotype of a RLFHF FH+/– cell, where no chromosome abnormality is seen as revealed for the karyotype of a control fibroblast at the 40th passage. (I) Electron microscope images of RLFHF FH+/– cells (4000× for both a and b), which are rich in mitochondria, rough endoplasmic reticulum and ribosomes. Western blot for FH protein expression in the 58th passage WT, 68th passage FH+/– and RLFHF cells is shown in (J) and corresponding histograms are shown in (K).